Identification and characterization of protease-resistant SecA fragments: secA has two membrane-integral forms.
We have identified and characterized the protease-resistant SecA fragments (X. Chen, H. Xu, and P. C. Tai, J. Biol. Chem. 271:29698-29706, 1996) through immunodetection with region-specific antibodies, chemical extraction, and sequencing analysis. The 66-, 36-, and 27-kDa proteolytic fragments in the membranes all start at Met1, whereas the 48-kDa fragment starts at Glu361. The overlapping of the sequences of the 66- and 48-kDa fragments indicates that they are derived from different SecA molecules. These two fragments were generated differently in response to ATP hydrolysis and protein translocation. Furthermore, the presence of membrane is required for the generation of the 48-kDa fragment but not for that of the 66-kDa fragment. These data suggest that there are two different integral forms of SecA in the membrane: SecA(S) and SecA(M). The combination of these two forms of SecA has several membrane-interacting domains. Both forms of SecA are integrated in the membrane, since both the 48- and 66-kDa fragments could be derived from urea- or Na2CO3-washed membranes. Moreover, all fragments are resistant to extraction with a high concentration of salt or with heparin, but the membrane-specific 48-kDa SecA domain is more sensitive to Na2CO3 or urea extraction. This suggests that this domain may interact with other membrane proteins in an aqueous microenvironment and therefore may form a part of the protein-conducting channel.
Chen
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Department of Biology, Georgia State University, Atlanta 30303, USA.
Brown
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Tai
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017421:Sequence Analysis; D013462:Sulfur Radioisotopes; D014316:Tritium; D014357:Trypsin / Q000378:metabolism*
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D013462:Sulfur Radioisotopes; D014316:Tritium; D014357:Trypsin; D019286:Endopeptidase K; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins